CHARACTERIZATION OF EXTRACELLULAR VESICLES RELEASED FROM MIXED CULTURES OF CHICK RETINA.

Introduction: Exosomes are small extracellular vesicles (EVs) (50–150nm) from an endocytic origin. They work as a mechanism of cargo diffusion of different molecules, such as microRNAs, that are a group of non-coding RNAs who acts by an interaction with mRNA targets interrupting the translation mechanism. They are found in different cells of the Central Nervous System (CNS), and interferes in different signaling pathways, such as adenosinergic system. Adenosine is a fundamental neuromodulator in several physiological and pathological events. Its acts through the interaction with its receptors: A1, A2A, A2B and A3 and its transport through the membrane is regulated by transmembrane proteins called equilibrative nucleoside transporters (ENTs), which were described in CNS into four isoforms, with ENT1/ENT2 being the predominant ones.

Aims: Characterize the profile of vesicles released by chick retinal cultures and investigate if miRNA-124 mimic regulates the ENT1 in this model.

Methods: All procedures were approved by the Ethics Committee on Animal Use of the UFF under protocol #1570300921. Mixed cultures of chick retina were obtained from embryos with eight days (E8) and through ultracentrifugation processes, we investigate if our cultures were able to produce exosomes, analyzing the vesicles sizes by Dynamic Light Scattering (DLS) and their morphology by transmission electron microscopy (TEM). We also transfected the mixed cultures with miR-124 at different concentrations (25, 50 and 100nM) for 24h or 48h. [3H]-Ado uptake assays were performed to measure ENTs activity and Western Blot to measure ENT1 protein levels.

Results: DLS analysis revealed that our extracellular vesicles have a size range of 94–155 nm, with an average size of 129.2±25.7nm (n=4). TEM revealed a spherical morphology of these vesicles (n=3). Exposure for 24 hours with miRNA-124 mimic did not induce any significant change in [3H]-Ado uptake (CT 100%; miR-124 25nM: 93,2±6,63 n.s.; miR-124 50nM: 92,62±5,03 n.s.; miR-124 100nM: 93,38±4,46 n.s. n=3) or protein levels of ENT1 (CT 100%; miR-124 25nM: 90,51±14,93 n.s.; miR-124 50 nM: 114,16±36,6 n.s.; miR-124 100nM: 102,81±37,89 n.s. n=3). However, a longer treatment for 48 hours induced a significant reduction in [3H]-Ado uptake at miR-124 at 100nM (CT 100%; miR-124 100nM: 80,50±6,37 *p<0,05. n=3) and a trend of decreasing ENT1 protein levels at miR-124 (50 and 100nM) conditions (CT 100%; miR-124 50nM: 82,85±14,21 n.s.; miR-124 100nM: 58,81±12,89 n.s. n=3).

Conclusions: These results suggest retinal exosomes as a possible source of microRNAs in this model and ENT1 as a potential regulatory target for miR-124.